Protein Synthesis

(i) Mechanism of ribosome recycling : Subsequent to the action of release factors at the step of termination, ribosomes remain bound to the mRNA in a post-termination complex. In eubacteria, the post-termination complexes are disassembled by the action of ribosome recycling factor (RRF) and elongation factor G (EFG). We have shown that specific interactions between RRF and EFG are essential to recycle the post-termination complexes. Such interactions between RRF and EFG are also required to recycle the stalled ribosomes (pre-termination ribosomal complexes) during the step of elongation by releasing peptidyl-tRNAs from them. The function of EFG in ribosome recycling is different from its classical role in translocation, and our recent observations suggest that a distinct set of interactions of EFG with RRF and the ribosome is crucial in this process. Further, we have shown that there is a functional interaction between RRF and IF3 in recycling of both the pre-, and post-termination ribosomal complexes. Present research is focused on the interplay of various factors during the various steps of protein synthesis.


A model for RRF action
A model for RRF action


(ii) The structure-function relationship of the E. coli initiator tRNA : Eubacterial initiator tRNAs (tRNAfMet) possess unique features such as a mismatch at the top of the acceptor stem and the three consecutive G, C base pairs in the anticodon stem. The latter feature which is highly conserved in all the three kingdoms of life has been implicated in preferential binding of tRNAfMet to the ribosomal P-site. How this feature is exploited by ribosomes in selecting tRNAfMet has been a long standing question. We have isolated several E. coli strains which allow initiation with tRNAs lacking the three consecutive G-C base pairs. In one of the strains, a severe deficiency of methionine and S-adenosyl-L-methionine; and lack of nucleoside methylations in rRNA allow initiation with tRNAfMet containing mutations in one, two or all the three G-C base pairs, and also with an elongator tRNA. Mutations in specific methyltransferases support a role for rRNA methylations in tRNAfMet selection on the ribosome. In yet another strain, reduction in levels of wildtype initiator tRNA allows initiation with a mutant tRNA lacking 3 G-C pairs, showing that there is a competition between initiator and elongator tRNAs for P-site binding. We have also shown that it is possible to sustain E. coli on a 3 G-C mutant tRNA and that the 3 G-C rule can in fact, be reduced to a middle G-C rule. Future studies are aimed at characterization of the remaining supressor strains to allow detailed understanding of the mechanism of initiation.


Clover leaf structure of initiator tRNA
Secondary structure of E.coli initiator tRNA


(iii) A single mammalian mitochondrial translation initiation factor functionally replaces two bacterial factors: In eubacteria, three initiation factors IF1, IF2, and IF3 are vital. We have shown that bovine mitochondrial IF2 (IF2mt) complements E. coli containing a deletion of IF2 gene (infB). We find that IF1 is no longer essential in an IF2mt-supported E. coli ΔinfB strain. We suggest that a conserved insertion of 37 amino acids in the IF2mt substitutes for the function of IF1. Deletion of this insertion from IF2mt supports E. coli for the essential function of IF2. However, in this background, IF1 remains essential. The future studies are aimed at understanding of how the 37 amino acid insert in IF2mt substitutes for the function of IF1.